Synthetically enveloped virus

ABSTRACT

A method of modifying a virus for in vivo delivery to a region of interest includes forming an enveloping composition including a lipid conjugate formed by conjugating at least one lipid with at least one hydrophilic compound via a linkage which is cleavable under conditions present in the region of interest and combining the virus with the enveloping composition to encompass the virus within the enveloping structure.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 62/313,270, filed Mar. 25, 2016, the disclosure of which isincorporated herein by reference.

GOVERNMENTAL INTEREST

This invention was made with government support under grant no. CA140215awarded by the National Institutes of Health. The government has certainrights in this invention.

BACKGROUND

The following information is provided to assist the reader inunderstanding technologies disclosed below and the environment in whichsuch technologies may typically be used. The terms used herein are notintended to be limited to any particular narrow interpretation unlessclearly stated otherwise in this document. References set forth hereinmay facilitate understanding of the technologies or the backgroundthereof. The disclosure of all references cited herein are incorporatedby reference.

Viral therapies or virotherapies are treatment regimens in whichbiotechnology is used to convert viruses into therapeutic agents byreprogramming the viruses to treat diseases. Currently, there are threeprimary branches of viral therapies including anti-cancer or oncolyticviruses, viral immunotherapy and viral vectors for gene therapy.

Rationally designed and engineered oncolytic viral (OV) therapies werefirst tested in the clinic over 25 years ago. However, it is only in thelast five years that clinical responses have begun to approach thepromise shown in pre-clinical models. The demonstration of enhancedresponses and/or survival seen in randomized clinical testing withseveral vectors, including talimogene laherparepvec (T-Vec),pexastimogene devacirepvec (Pexa-Vec) and coxsackievirus A21 (CVA21)indicated that US Food and Drug Administration (FDA) approval willlikely be forthcoming for one or more vectors in different indications.The administration of T-Vec for metastatic melanoma has, for example,recently been approved by the US FDA. However, all of those trials haverelied on direct intratumoral injection. Disseminated disease is themajor cause of cancer-related death and cannot be adequately treatedwith intratumoral injections. In addition, because an initial round oftreatment raises an immune response against the therapy itself,subsequent cycles or treatment are further limited in their ability toachieve systemic delivery. Although the possibility for systemic OVdelivery, even leading to remission of disseminated disease, has beendemonstrated in the clinic, such case reports merely act to highlightthe future potential of the field if reliable and reproducible systemicdelivery could be achieved.

A variety of different approaches have been proposed to try to overcomelimitations in systemic treatment via an OV or other viral vector.Approaches involving immunosuppression of the cancer patient havelargely been abandoned as it has become clear that the immunotherapeuticeffects of OV vectors are an important component to their tumor-killingpotential. Sequential use of related but serologically distinct vectorsand the use of pre-infected cells as delivery vehicles have met withsome success, but add to the complexity and cost of the therapy. Use ofintratumoral or local-regional delivery can be used in some limitedsettings, but typically fail to treat widespread metastatic disease.Even if the OV therapy is capable of raising an adaptive immune responsetargeting tumor antigens, metastases are often antigenicaly distinctfrom the primary tumor.

Approaches that involve chemical modifications of the viral particleitself show theoretical promise. Such modifications involve directchemical attachment of large inert molecules (such as PEG) to the viralparticle, or the addition of a lipid envelope or polymer-based coatingaround the particle. Although a number of such approaches havedemonstrated the capacity to protect the viral particle and/or detargetthe virus from natural target tissues (particularly the liver), suchapproaches commonly disrupt the virus's evolved pathways of cell entryand thereby limit the ability of the virus to infect tumor cells. Theuse of cationic polymers that are pH sensitive may mitigate thislimitation, but have raised toxicity concerns. Although there is a greatneed for technologies in which the viral particle is modified, none haveadvanced into a clinical setting to date.

SUMMARY

In one aspect, a method of modifying a virus for in vivo delivery to aregion of interest includes forming an enveloping composition includinga lipid conjugate formed by conjugating at least one lipid with at leastone hydrophilic compound via a linkage which is cleavable underconditions present in the region of interest and combining the viruswith the enveloping composition to encompass the virus within thestructure. The enveloping composition may, for example, form a lipidbilayer to encompass/envelope the virus.

In a number of embodiments, the at least one lipid is selected from thegroup consisting of n-docosanoic acid, arachidic acid, stearic acid,palmitic acid, myristic acid, lauric acid, oleyl acid, vitamin E,embelin, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, or acompound having the formula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof. In a number ofembodiment, the at least one lipid has the formula:

The at least one lipid may, for example, be selected from the groupconsisting of S-trans, trans-farnesylthiosalicylic acid, S-trans,trans-farnesylthiosalicylic acid amide (FTS-amide), S-trans,trans-farnesylthiosalicylic acid methylamide (FTS-MA) and S-trans,trans-farnesylthiosalicylic acid dimethylamide (FTS-DMA). In a number ofembodiments, the at least one lipid is farnesylthiosalicylic acid or afarnesylthiosalicylic acid amide.

In a number of embodiments, a family of the virus is selected from thegroup consisting of poxvidrae, denoviridae, herpesviridae,picornaviridae, rhabdoviridae, paramyxoviridae, retroviridae,togaviridae or reoviridae. The virus may, for example, be selected fromthe group consisting of a vaccinia virus, a myxoma virus, an avipoxvirus, an adenovirus, a herpes simplex virus (HSV) coxsackie virus, avesicular stomatitis virus (VSV), a Newcastle disease virus (NDV), anadeno-associated virus (AAV), a polio virus, a lenti virus, aretrovirus, a reovirus, or a sindbis virus. In a number of embodiments,the family of the virus is poxvidrae. The virus may, for example, be avaccinia virus. The virus may, for example, be a mature vaccinia virus.

In a number of embodiments, the region of interest includes a tumor, andthe virus is modified to treat the tumor.

In a number of embodiments, the at least one hydrophilic compoundincludes at least one hydrophilic oligomer or at least one hydrophilicpolymer. The hydrophilic oligomer or the hydrophilic polymer may, forexample, be selected from the group consisting of a polyalkylene oxide,a polyvinylalcohol, a polyacrylic acid, a polyacrylamide, apolyoxazoline, a polysaccharide and a polypeptide. In a number ofembodiments, the at least one hydrophilic compound is a polyalkyleneoxide. The polyalkylene oxide may, for example, be a polyethyleneglycol. A polyethylene glycol or other hydrophilic polymer may, forexample, have a molecular weight of at least 1 KDa.

In a number of embodiments, the linkage is sensitive to pH. The linkagemay, for example, include a hydrazine group.

The enveloping composition may include at least one co-lipid. The atleast one co-lipid may, for example, be a phospholipid. The method mayfurther include providing an additive in the enveloping composition. Theadditive may, for example, increase or decrease stability of theenveloping composition. Cholesterol may, for example, be included as anadditive. DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) is afusogenic lipid, which may be used to decrease the lipid stability andfacilitate the release of virus.

In another aspect, a formulation (for example, for in vivo delivery to aregion of interest) includes a virus, a synthetic enveloping composition(as described above) encompassing the virus and including a lipidconjugate formed by conjugating at least one lipid with at least onehydrophilic compound via a linkage which is cleavable under conditionspresent in the region of interest. As described above, the envelopingcomposition may a lipid bilayer encompassing the virus.

In another aspect, a method of in vivo delivery of a virus to a regionof interest includes injecting a formulation as described above.

In another aspect, a method of modifying a virus for in vivo delivery toa region of interest includes forming an enveloping composition includesa lipid having the formula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof, and combining thevirus with the enveloping composition to encompass the virus. Theenveloping composition may from a lipid bilayer. The lipid may, forexample, be selected from the group consisting of S-trans,trans-farnesylthiosalicylic acid, S-trans, trans-farnesylthiosalicylicacid amide (FTS-amide), S-trans, trans-farnesylthiosalicylic acidmethylamide (FTS-MA) and S-trans, trans-farnesylthiosalicylic aciddimethylamide (FTS-DMA).

The enveloping composition forms a lipid bilayer. In a number ofembodiments, the lipid is selected from the group consisting of S-trans,trans-farnesylthiosalicylic acid, S-trans, trans-farnesylthiosalicylicacid amide (FTS-amide), S-trans, trans-farnesylthiosalicylic acidmethylamide (FTS-MA) and S-trans, trans-farnesylthiosalicylic aciddimethylamide (FTS-DMA).

The enveloping composition may, for example, include at least oneco-lipid. The at least one co-lipid may, for example, be a phospholipid.The method may further include providing an additive in the envelopingcomposition. The additive may, for example, increase or decreasestability of the enveloping composition. Cholesterol may, for example,be included as an additive.

In a further aspect, embodiment a formulation (for example, for in vivodelivery to a region of interest) includes a virus, a syntheticenveloping composition encompassing the virus and comprising a lipidhaving the formula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof. The envelopingcomposition may be further characterized as described above.

In still a further aspect a composition is formed by conjugating atleast one lipid with at least one hydrophilic compound via apH-sensitive hydrazine linkage which is cleavable under conditionspresent in the region of interest. The composition may, for example,form a lipid bilayer. The at least one lipid may, for example, beselected from the group consisting of n-docosanoic acid, arachidic acid,stearic acid, palmitic acid, myristic acid, lauric acid, oleyl acid,vitamin E, embelin, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol,or a compound having the formula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof. In a number ofembodiments, the at least one lipid has the formula:

The at least one lipid may, for example, be selected from the groupconsisting of S-trans, trans-farnesylthiosalicylic acid, S-trans,trans-farnesylthiosalicylic acid amide (FTS-amide), S-trans,trans-farnesylthiosalicylic acid methylamide (FTS-MA) and S-trans,trans-farnesylthiosalicylic acid dimethylamide (FTS-DMA). In a number ofembodiments, the at least one lipid is farnesylthiosalicylic acid or afarnesylthiosalicylic acid amide (or a biologically active derivativethereof).

The at least one hydrophilic compound may, for example, include at leastone hydrophilic oligomer or at least one hydrophilic polymer. Thehydrophilic oligomer or the hydrophilic polymer may, for example, beselected from the group consisting of a polyalkylene oxide, apolyvinylalcohol, a polyacrylic acid, a polyacrylamide, a polyoxazoline,a polysaccharide and a polypeptide. In a number of embodiments, the atleast one hydrophilic compound is a polyalkylene oxide. The polyalkyleneoxide may, for example, be a polyethylene glycol. The polyethyleneglycol or other hydrophilic polymer may, for example, be a molecularweight of at least 1 KDa. In a number of embodiments, the linkage issensitive to pH.

The synthetic lipid-derived liposomal envelopes or envelopingcompositions hereof create an enveloped virus with dramatically improvedtherapeutic potential and novel properties. The synthetically envelopedvirus retains (and in some cases even increases) infectivity of thevirus over non-enveloped virus. The synthetically enveloped virus mayincrease absolute infection (as determined by viral gene expression) ina target tissue (tumor) in vivo relative to a non-enveloped virus.

Delivery of synthetically enveloped virus to tumors via an intravenousroute may be inefficient as a result of its rapid clearance by thereticuloendothelial system (RES). This problem may be resolved, however,through covalent attachment of hydrophilic chemical structures, whichmay, for example, be inert, (such as polyethylene glycol) to the lipidenvelope, using bonds that may be cleaved under particular environmentalconditions (such as low pH, REDOX potential, presence of proteasesetc.). As used herein, the term “inert” refers to hydrophilic chemicalstructures (for example, polymers) that do not have significantbiological effects other than the intended shielding effect. In thismanner, the viral vectors may be further detargeted and protected whilein circulation. Release of the synthetic enveloped virus from thehydrophilic outer shell can be made conditional on the targetmicroenvironment (such as low pH typically found in the tumor, increasedoxidative stress found in inflammation etc.). Addition of othertargeting factors into the synthetic may also be used to target thevirus to certain cell types.

The synthetically enveloped viruses hereof do not require specificprotein-protein interactions or covalent bonds to retain the outerenvelope. As a result, the synthetic envelope is stable but retains thecapacity to be shed during an infection step. This is in contrast to,for example, the natural lipid envelope added to the MV virus to formthe EV form. In this case the outer membrane requires specific proteininteractions and is still highly unstable. In that regard, it hastraditionally been impossible to purify and store the EV form ofvaccinia because the outer envelope is too unstable. Using the syntheticenvelopes hereof, a composition may be stored for at least some period(freeze/thawed, etc). Nonoptimized studies have shown that thecompositions may be stored for at least one month with one freeze/thawcycle.

The present devices, systems, methods and compositions, along with theattributes and attendant advantages thereof, will best be appreciatedand understood in view of the following detailed description taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an idealized, schematic representation of theformation of a representative embodiment of enveloping a virus whereinthe envelope is a lipid bilayer.

FIG. 1B illustrates representative examples of cleavable groups for usein the envelopes hereof.

FIG. 1C illustrates representative examples of lipids for use in theenvelopes hereof.

FIG. 1D illustrates viral gene expression and anti-tumor effects afterintravenous delivery in naïve or immunized mice, wherein BALB/c micewere either immunized (IP injection of 1e6 plaque-forming units or PFUof wild type vaccinia strain WR) or not, and 28 days later implantedsubcutaneously with 4T1 tumors.

FIG. 1E illustrates viral gene expression and anti-tumor effects afterintratumoral delivery in naïve or immunized mice, wherein BALB/c micewere either immunized (intraperitoneal or IP injection of 1e6 PFU ofwild type vaccinia Western Reserve strain or WR) or not, and 28 dayslater implanted subcutaneously with 4T1 tumors.

FIG. 1F illustrates tumor volume as a function of time after intravenousdelivery in naïve or immunized mice.

FIG. 1G illustrates tumor volume as a function of time afterintratumoral delivery in naïve or immunized mice.

FIG. 2A illustrates an electron microscope photomicrograph confirmingviral encapsulation, wherein virus (WR.TK-Luc+) was enveloped with alipid layer including PEG-FTS-H, pH-sensitive lipid and encapsulationwas confirmed by electron microscopy (EM) and zetasizer.

FIG. 2B illustrates another electron microscope photomicrographconfirming viral encapsulation with a lipid that was not pH sensitive.

FIG. 2C illustrates a graph of size distribution by intensity for thevirus prior to enveloping.

FIG. 2D illustrates a graph of size distribution by intensity for virus(WR.TK-Luc+) enveloped with a lipid layer including PEG-FTS-H lipid.

FIG. 3 illustrates in vitro comparison of different coatingformulations, wherein Virus (WR.TK-Luc+) was enveloped with eitherPEG-FTS or DSPE-PEG containing lipid layers, or left uncoated.

FIG. 4A illustrates in vivo delivery of naked and enveloped virus,wherein athymic nu/nu mice were implanted with HCT 116 tumors and oncepalpable, high dose vaccinia immune globulin or VIG was delivered via IPinjection 24 h prior to IV delivery of naked or enveloped WR.TK-Luc+(1e8 PFU/mouse, n=4/group), and viral gene expression was measured asbioluminescence 24 h later.

FIG. 4B illustrates viral expression as bioluminescence for in vivodelivery of naked and enveloped virus.

FIG. 5A illustrates viral gene expression measured after 24 h of tumorgrowth as bioluminescence in fully immunized mice, wherein C57/BL6 micewere immunized (or not) 28 days prior to implantation with MC38 tumorcells; and wherein once tumors were palpable mice were treated with anIV injection of WR.TK-Luc+, naked or enveloped as before.

FIG. 5B illustrates anti-tumor effect as measured by tumor volume (viacaliper measurement) over time in the studies of FIG. 5A.

FIG. 6A illustrates the effects of repeat delivery in tumor-bearing miceon tumor volume (via caliper measurement), wherein mice (BALB/c bearingsubcutaneous Renca tumors) were treated with an IV dose of WR.TK-.GFP+and then, with three days between treatments, with WR.TK-Luc+.

FIG. 6B illustrates the effects of repeat delivery in tumor-bearing miceon tumor volume (via caliper measurement), wherein mice (BALB/c bearingsubcutaneous Renca tumors) were treated with an IV dose of WR.TK-.GFP+and then, with 17 days between treatments, with WR.TK-Luc+.

FIG. 7 illustrates percent infection rate of several types of virusesenveloped or encapsulated under the methods hereof as compared to suchviruses without the envelopes hereof.

FIG. 8 illustrates the structure of a number of pH-sensitive conjugateshereof.

DETAILED DESCRIPTION

It will be readily understood that the components of the embodiments, asgenerally described and illustrated in the figures herein, may bearranged and designed in a wide variety of different configurations inaddition to the described representative embodiments. Thus, thefollowing more detailed description of the representative embodiments,as illustrated in the figures, is not intended to limit the scope of theembodiments, as claimed, but is merely illustrative of representativeembodiments.

Reference throughout this specification to “one embodiment” or “anembodiment” (or the like) means that a particular feature, structure, orcharacteristic described in connection with the embodiment is includedin at least one embodiment. Thus, the appearance of the phrases “in oneembodiment” or “in an embodiment” or the like in various placesthroughout this specification are not necessarily all referring to thesame embodiment.

Furthermore, described features, structures, or characteristics may becombined in any suitable manner in one or more embodiments. In thefollowing description, numerous specific details are provided to give athorough understanding of embodiments. One skilled in the relevant artwill recognize, however, that the various embodiments can be practicedwithout one or more of the specific details, or with other methods,components, materials, et cetera. In other instances, well knownstructures, materials, or operations are not shown or described indetail to avoid obfuscation.

As used herein and in the appended claims, the singular forms “a,” “an”,and “the” include plural references unless the context clearly dictatesotherwise. Thus, for example, reference to “a virus” includes aplurality of such viruses and equivalents thereof known to those skilledin the art, and so forth, and reference to “the virus” is a reference toone or more such viruses and equivalents thereof known to those skilledin the art, and so forth. Recitation of ranges of values herein aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range. Unless otherwiseindicated herein, and each separate value, as well as intermediateranges, are incorporated into the specification as if individuallyrecited herein. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontraindicated by the text.

The terms “virus”, “virion” and “viral particle” are usedinterchangeably herein. Often, the term “virus” is used collectively. Avirus is a submicroscopic infectious agent that is unable to grow orreproduce outside a host cell. A virus includes genetic material (DNA orRNA) within a protective protein coat known as a capsid. Capsid shapesvary from simple helical and icoshedral (polyhedral or near-spherical)forms, to more complex structures with tails or an envelope. Virusesused in the systems, methods and compositions hereof may be naturalviruses or engineered/modified viruses.

As used herein, the term “polymer” refers to a chemical compound that ismade of a plurality of small molecules or monomers that are arranged ina repeating structure to form a larger molecule. Polymers may occurnaturally or be formed synthetically. The use of the term “polymer”encompasses homopolymers as well as copolymers. The term “copolymer” isused herein to include any polymer having two or more differentmonomers. Copolymers may, for example, include alternating copolymers,periodic copolymers, statistical copolymers, random copolymers, blockcopolymers, graft copolymers etc. Examples of polymers include, forexample, polyalkylene oxides.

As used herein, the term “lipid” refers to a group of molecules thatinclude, for example, fats, fatty acids, waxes, sterols, fat-solublevitamins (such as vitamins A, D, E, and K), monoglycerides,diglycerides, triglycerides, phospholipids, and others. Lipids arerelated by their solubility in nonpolar organic solvents and generalinsolubility in water. Phospholipids are a class of lipids that, forexample, form a major component of all cell membranes. Phospholipids mayform lipid bilayers as a result of their amphiphilic characteristic. Aphospholipid molecule may, for example, include two hydrophobic fattyacid “tails” and a hydrophilic “head” joined together by a glycerolmolecule.

Efforts to protect oncolytic viruses with lipid envelopes or polymercoatings may be successful at detargeting the viral vectors from normaltissues, such as the liver, and even evading anti-viral immunity to someextent. However this benefit has traditionally come at the cost of asignificant loss of viral infectivity of the tumor (with recovery ofaround 5% of the virus typical). Although cationic polymers may exhibitbetter retention of viral infectivity than other encapsulationmaterials, cationic polymer exhibit toxicity issues. Strategies of viralencapsulation hereof overcome many of the limitations associated withexisting methodologies and may provide a powerful means to deliver virusto tumor targets, even in the face of pre-existing anti-viral immunity.Viral encapsulation systems, methods and compositions hereof arediscussed in connection with representative examples of oncolyticviruses. However, one skilled in the art will recognize that such viralencapsulation systems, methods and compositions are applicable in anyviral therapy.

As described above, strategies of viral encapsulation hereof overcomemany of the limitations associated with existing methodologies andprovide a powerful mechanism to deliver virus to tumor targets, even inthe face of pre-existing anti-viral immunity. In a number ofembodiments, viruses, virions or viral particles (collectively, viruses)are enveloped or encapsulated in a synthetic envelope formed from acomposition including a hydrophobic/lipid domain and a hydrophilicdomain linked to the hydrophobic/lipid domain. The hydrophilic domainmay, for example, be separable from the hydrophobic/lipid domain underphysiological conditions present in a target region or region ofinterest (that is, a region targeted for treatment). There may be morethan one region of interest distributed throughout the body (forexample, in the case of disseminated cancerous tumors). In a number ofembodiments, the synthetically enveloped virus is a synthetic version ofa naturally occurring and infectious enveloped viral form of the virus.In such embodiments, infection of target cells may, for example, beoptimized.

Synthetic envelops hereof may, for example, include a synthetic lipidconjugate including a hydrophobic/lipid domain linked or conjugated to ahydrophilic domain (for example, a hydrophilic polymer domain including,for example, a polyethylene oxide such as polyethylene glycol) via alinker which is labile or cleavable under physiological conditionspresent in the region of interest. In a number of embodiments, thelinker is a pH sensitive linker. Such a modified virus, is stable inblood and can evade anti-viral antibodies, thereby allowing systemicdelivery to, for example, an acidic tumor environment. Once, in thetumor, the hydrophilic domain is de-grafted via cleavage of thepH-sensitive linker, destabilizing the synthetic virus envelope andleading to release of the infectious synthetically enveloped virus. In anumber of representative studies hereof, such a synthetically envelopedvirus displayed enhanced systemic delivery and therapeutic effects inmouse models, even in the face of pre-existing anti-viral immunity orduring repeated systemic delivery.

FIG. 1A illustrates an idealized schematic diagram of a representativeembodiment of a synthetic lipid envelope hereof encompassing a virus. Inthe illustrated embodiment, the envelope forms a closed lipid bilayer. Abilayer is a preferred structure of lipids in aqueous solutions. In anumber of embodiments hereof, the synthetic lipid conjugates hereof arecombined with one or more co-lipids in forming the synthetic envelope.The colipid(s) may, for example, form the major lipid component of theenvelop/bi-layer. In a number of embodiments, co-lipids in the form ofphospholipids are the major component in the envelope/lipid bilayer.Increasing the amounts of synthetic lipid conjugates helps to decreasethe interaction with blood components and the rapid clearance from thecirculation. However, incorporation of too much such lipid conjugatesmay compromise the stability of the lipid bilayer. In a number ofembodiments hereof, no greater than 20 mol % of hydrophiliccompound-lipid conjugates are incorporated. The hydrophiliccompound-lipid conjugates may, for example, be present in the range of 5to 20 mol %. Co-lipids may, for example, be present in the range of 40to 95 mol %. Additives such as DOPE or cholesterol may, for example, bepresent in the range of 0 to 40 or 10 to 40 mole %. Variousphospholipids maybe be used as co-lipids herein including, but notlimited to, natural and synthetic phosphatidylcholines or PC (forexample, L-α-phosphatidylcholine), phosphatidylethanolamine or PE (forexample, L-α-phosphatidylethanolamine) and phosphatidylinositol or PI(for example, L-α-phosphatidylinositol). Natural phosphatidylcholinesinclude, for example, egg PC, heart PC, soy PC, brain PC and liver PC.Synthetic phosphatidylcholines include, for example,1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Naturalphosphatidylethanolamines include, for example, egg PE, heart PE, soyPE, brain PE, liver PE. Synthetic phosphatidylethanolamines include, forexample, 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), and1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). Naturalphosphatidylinositols include, for example, liver PI, soy PI, brain PI.Synthetic phosphatidylinositols include, for example,1,2-dihexadecanoyl-sn-glycero-3-phospho-(1′-myo-inositol) DPPI,1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol) DOPI, and1,2-distearoyl-sn-glycero-3-phospho-(1′-myo-inositol) (DSPI). Theefficiency of the enveloped virus may, for example, further tuned byreadily optimizing the lipid composition of the envelope/bilayer for aparticular envelope, virus and/or application.

The synthetic envelopes hereof may, for example, include one or morefurther substituents which are references generally as additives in FIG.1A. Such additives may, for example, function to modulate the stabilityof the synthetic envelope. Cholesterol, for example, may assist instabilizing a lipid bilayer. On the other hand,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is a fusogeniclipid. Inclusion of DOPE may decrease the lipid stability and facilitatethe release of virus from endosome following intracellular delivery.Various other additive may be similarly used to adjust or tune theproperties of the compositions or formulations hereof,

The identity and concentration of various additives is also subject tooptimization for a particular envelope, virus and/or application viaknown activities and/or routine experimentation. In the case of astabilizing component such as cholesterol, for example, while a stablelipid bilayer might be desirable, an overly stable bilayer maynegatively affect the un-coating of virus following internalization intocells.

As discussed above, synthetic lipid envelopes (including synthetic lipidconjugates hereof) assist in prevent nonspecific interaction of viruseswith serum proteins, which is important for a prolonged circulation timein the blood and effective targeting to the tumors. Further, the labileor cleavable linker in the synthetic lipid conjugates hereof providesfor controlled shedding of the synthetic envelope.

In a number of studies, representative vaccinia viruses wereencapsulated or enveloped with synthetic envelopes hereof. The vacciniavirus, which may be the backbone for multiple clinical vectors, canexist in different infectious forms, distinguished by the number oflipid envelopes shrouding the viral core. These forms include the mostbasic infectious form (Intracellular Mature Virus, IMV or Mature Virus,MV) and a version of the IMV that has an additional host cell derivedlipid envelope (the Enveloped virus, EV). The application of anartificial or synthetic hydrophobic/lipid envelope to the IMV form ofthe virus creates a synthetic version or mimic of the EV form that hasnaturally evolved multiple routes of cell entry. Unlike a number ofartificially enveloped or coated viruses, synthetic EV may retain a morerobust capacity to infect tumor cells. In a number of representativeembodiments, use of a pH-sensitive linker to attach a hydrophilic domainto the hydrophobic/lipid envelope acts to protect the viral particlewhile in circulation, but is cleaved from the envelope encompassing theviral particle once in the acidic tumor environment or after entry intothe endosomal pathway. In the case of target regions or regions ofinterest other than acidic tumor environments, the cleavable bond may beresponsive or sensitive to other microenvironments present within theregions of interest (for example, reduction/oxidation potential, hypoxiaand matrix metalloproteinase-9) (see FIG. 1C for representativeexamples). The combination of synthetically mimicking a naturallyexisting viral form and incorporation of advanced lipid technology ispresent in a number of embodiments hereof. However, the systems, methodsand compositions hereof are applicable to viral therapies generally andneed not mimic a naturally existing enveloped viral form.

As described above, the incorporation of a vaccinia backbone in a numberof embodiment hereof allows the addition of a synthetic lipid envelopeto a form of the virus (the IMV) resulting in the production of asynthetic version or mimic of a naturally evolved form of the virus(EV). In this way, a synthetic version of a naturally occurring virus isformed and the viruses natural cell entry pathways are not disrupted.Indeed, even when a ‘standard’ lipid envelope (that is, a lipid envelopnot including a synthetic lipid conjugate hereof) is used, recovery ofthe vaccinia virus is 10 times better than reported in the previousmodels.

The hydrophilic compounds used in forming the hydrophilic domains of thesynthetic lipid conjugates hereof may, for example, include at least onehydrophilic oligomer or at least one hydrophilic polymer. Thehydrophilic oligomer or the hydrophilic polymer may, for example, beselected from the group consisting of a polyalkylene oxide, apolyvinylalcohol, a polyacrylic acid, a polyacrylamide, a polyoxazoline,a polysaccharide and a polypeptide. In a number of embodiment, the atleast one hydrophilic compound is a polyalkylene oxide. In a number ofrepresentative embodiments hereof, the polyalkylene oxide is apolyethylene glycol (PEG). In a number of embodiments, the hydrophiliccompound (for example, PEG) has a molecular weight of at least 1 KDa(for example, in the range of approximately 1 KDa to 10 KDa). In anumber of embodiments, the hydrophilic domain has a molecular weight inthe range of approximately 1 KDa to 5 KDa. The hydrophilic domain(s) ofthe synthetic lipid conjugates hereof, for example, include a single ormultiple chains.

Many different hydrophobic compounds may be used in the hydrophobicdomains of the synthetic lipid conjugates hereof. Representative exampleof such hydrophobic compounds are provided in FIG. 1B. The hydrophobicdomain(s) of the synthetic lipid conjugates hereof, for example, includea single or multiple chains.

In a number of representative embodiments, synthetic lipid conjugatecompositions used to create the envelope included farnesylthiosalicylicacid (FTS) or a derivative of farnesylthiosalicylic acid conjugated toPEG with a pH sensitive and tunable hydrazine linker. The use of suchmaterials provides several advantages. The FTS or FTS derivative may,for example, be a biologically active derivative offarnesylthiosalicylic acid including, for example, be S-trans,trans-farnesylthiosalicylic acid, S-trans, trans-farnesylthiosalicylicacid amide, S-trans, trans-farnesylthiosalicylic acid methylamide(FTS-MA) or S-trans, trans-farnesylthiosalicylic acid dimethylamide(FTS-DMA). Other biologically active farnesylthiosalicylic acidderivatives are also suitable for use herein. In that regard, FTS itselfhas antitumor activity (as a ras inhibitor) that can combine with thedifferent mechanisms of anti-tumor activity brought through the viralpayload. Moreover, the cleavage of PEG-FTS-H in response to pH changesmay be readily fine-tuned by, for example, choosing various lengths ofthe carbon chain or appropriate electron-withdrawing/contributing groupsclose to the hydrazone linker. This methodology provides control overthe rate of PEG degrafting following the delivery of enveloped virusesto the tumor tissues.

S-trans, trans-farnesylthiosalicylic acid (FTS), which is shown below,

is a synthetic farnesylcysteine mimetic that acts as a potent andespecially nontoxic Ras antagonist. Constitutively active Ras caused bymutation in the Ras family of proto-oncogenes is present in one-third ofhuman cancers, with the highest incidence of mutational activation ofRas being detected in pancreatic (90%) and colon (50%) cancers. Ras isalso activated in cancer cells by other mechanisms. In particular,hyperactivation of the epidermal growth factor receptor (EGFR) tyrosinekinase activity causes persistent activation of Ras and Ras-mediatedsignaling. The activated form of Ras constitutively activates itsdownstream effectors, contributing to cell transformation. FTS caninhibit both oncogenically activated Ras and growth factorreceptor-mediated Ras activation, resulting in the inhibition ofRas-dependent tumor growth. FTS can inhibit Ras transforming activityand reverse the transformed phenotype of Ras-transformed fibroblasts.FTS has demonstrated significant reduction of Ras levels in a wide arrayof established cancer models and inhibition of tumor growth in animalswith no adverse toxicity. One major mechanism involves affectingmembrane interaction of Ras by competing with Ras for binding toRas-escort proteins, facilitating its degradation, and thus disruptingRas protein to signal in the plasma membrane. In addition to itsantitumor activity, FTS also exhibits anti-inflammatory activity.Conjugation of FTS or an FTS derivative having, for example, theformula:

Wherein R₁, R₂, R₃, R₄, R₅, X and Z are defined as describe above withone or more hydrophilic compounds (for example, hydrophilic oligomers orpolymers such a polyethylene glycol or PEG) may provide antitumor or Rasantoginst activity independent of and synergistic with the viraltherapy.

In a number of representative embodiments hereof, pH-sensitivecompositions were formed by conjugating a hydrophilic PEG segment to oneor more hydrophobic FTS-hydrazide or FTS-H segments with a cleavablehydrazine linker. The use of a representative PEG-(FTS-H)₂ (pH sensitivePEG linker) on the lipid envelope resulted in 100% recovery ofinfectious vaccinia virus in tissue culture, which has not beenpreviously achieved with other encapsulation technologies. The envelopealso provides protection against neutralizing antibody, confirming thatthe envelope is active and functioning to protect the virus as expected.

Further, in vivo applications in mouse tumor models showed that use ofenveloped vaccinia viruses hereof not only reduced viral uptake innon-tumor tissues, but actually resulted in improved/increased deliveryto the tumor (relative to naked virus in naïve mice). This is the firsttime of which the inventors are aware that an encapsulation technologyactually enhanced delivery to the tumor.

Delivery of an active virus to a tumor or other region of interest isonly possible in the face of anti-viral immunity when the viral vectorswere enveloped. In a number of embodiments hereof, enveloped virusdelivered systemically in fully immunized mice actually displayedincreased viral gene expression from the tumor compared to naked virusdelivered in naïve (non-immunized) mice. This is a dramatic improvementon any previously reported approach.

Enhanced delivery of the enveloped viruses hereof also manifested itselfin enhanced therapeutic activity in several manners. In that regard,enveloped virus displayed enhanced therapeutic effects relative to nakedvirus when either was delivered systemically in naïve animals. Indeed,the therapeutic benefit achieved when enveloped virus was deliveredsystemically in fully immunized mice was even better than for nakedvirus in naïve, non-immunized mice (whereas naked virus in immunizedmice had no therapeutic effect). Moreover, when repeat cycles oftreatment were applied in a naïve, tumor-bearing animal, additionalcycles had no additional therapeutic benefit for naked virus, butprovided significant further benefit when enveloped virus was used. Suchdata indicate that systems, methods and compositions hereof provide asignificant advance over other reported approaches.

In a number of representative experiments, the capacity for delivery ofnaked virus (determined by luciferase transgene expression within thetumor) and therapeutic outcome after delivery via different routes andwith or without pre-existing immunity were explored. BALB/c mice wereeither immunized (IP injection of 1e6 PFU of wild type vaccinia strainWR) or not, and 28 days later implanted subcutaneously with 4T1 tumors.Once large tumors were formed (that is tumors having a volume ofapproximately 300-400 mm³), mice were treated with either intravenous(tail vein) or intratumoral injection of a model oncolytic vacciniastrain (1e8 PFU of strain WR with a deletion in the thymidine kinase, TKgene and expressing luciferase as a reporter; all three vaccinia strainscurrently undergoing clinical testing contain a deletion in the TKgene.) Subsequent tumor volume and viral gene expression from within thetumor were followed as set forth in FIG. 1D through 1G.

For naive mice, there were no significant differences in anti-tumoreffects between intravenous or intratumoral delivery. Although viralgene expression appeared slightly lower after intravenous delivery thiswas not significant (p=0.1). In previously immunized mice, intravenousdelivery resulted in almost no viral gene expression in the tumor(background levels of bioluminescence were determined at 1e4ph/sec/tumor). Unsurprisingly, this correlated with no therapeuticbenefit. Intratumoral delivery in previously immunized mice did producedetectable viral gene expression from the tumor, but this wasstill >50-fold less than viral gene expression for naïve mice. Withoutlimitation to any mechanism, the reduction may largely be a result ofanti-viral T-cell based immunity targeting infected tumor cells, asimaging was taken 24 h post delivery, and prior to spread of progenyvirus. Notwithstanding the significant reduction in viral geneexpression, there is actually a significant increase in therapeuticeffect. The increase in therapeutic effect may, for example, be mediatedby the immunotherapeutic effect of the OV therapy (as oncolytic effectsare reduced). Those results both reinforce the hypothesis that the mosteffective OV therapies act primarily as immunotherapies, but alsohighlight the potential importance of successful OV delivery to thetumor in pre-immunized patients.

Lipid-hydrophilic polymer conjugates or composition such aslipid-alkylene oxide conjugates, may be used in forming micelles ascarriers for delivery of small-molecule chemotherapies to tumor targets.Incorporation of an additional labile linkage such as a reductionsensitive (for example, disulfide) linkage into a lipid-hydrophilicpolymer conjugate such a conjugate of 5 kDa PEG and twofarnesylthiosalicylic acid groups (PEG5k-FTS₂) led to an increase intumor cell growth inhibitory effect and a further improvement in itsperformance in delivery of, for example, paclitaxel (PTX) to tumor cellsin vitro and in vivo. See, for example, U.S Patent ApplicationPublication Nos. 2015/0306034 and 2015/0231271, the disclosures of whichare incorporated herein by reference. Synthetic techniques thereof maybe adapted for use in the synthesizing lipid-hydrophilic conjugateshereof.

As described above, in a number of representative embodiments hereof,pH-sensitive compositions were formed by conjugating a hydrophilic PEGsegment to one or more hydrophobic FTS-H segments with a cleavablelinker such as a hydrazine linker. Once again, the stability of thehydrazine linker may be readily modulated by choosing different carbonchain lengths or appropriate electron-withdrawing/contributing groupsaround the hydrazine linker. It is thereby possible to develop a linkerthat is cleaved when exposed to the acidic pH found in, for example, atumor microenvironment.

In a representative methodology, lipid films containing DMPC:Cholesterol: PEGSK-FTS-H2 at a 2:1:0.1 ratio were mixed with the MatureVirus (MV form of oncolytic vaccinia WR.TK-.Luc+) in a PBS buffer andsonicated to create lipid enveloped viral particles. The viralpreparation methods produce virus containing >98% MV, which contains asingle outer envelope (as opposed to the Enveloped virus, EV form thathas an additional lipid envelope). The synthetically enveloped MV wereexamined by EM to confirm that close to 100% of the viral particles wereenveloped, and that viral particles were enveloped as single particles(with no clumps or doublets) (see FIGS. 2A and 2B). In addition,electron microscopy was used to confirm the integrity of the envelopes.FIGS. 2C and 2D illustrates size distribution by intensity for virusprior to enveloping and for virus (WR.TK-Luc+) enveloped with a lipidlayer including PEG-FTS-H lipid, respectively.

In a number of initial in vitro experiments, MV vaccinia virus was againencapsulated with a PEGSK-FTS-H₂ containing envelope. The experimentalresults were compared to naked virus and a more standard lipidencapsulation technology (that is, a lipid encapsulation technologyother than the those including the synthetic lipid conjugates hereofand, thus, not including a cleavable hydrophilic domain) DMPC:Cholesterol: DSPE-PEG2K at a 2:1:0.1 M ratio) so that the advantages ofencapsulating the MV form of vaccinia and the use of a pH-sensitiveenvelope encapsulation hereof could be explored.

Naked virus, PEG-FTS and DSPE-PEG enveloped virus were mixed with highdoses of VIG (vaccinia immunoglobulin) or PBS for 30 minutes beforeaddition to a fresh cell layer (of HeLa cells). The PEG-FTS envelopedvirus groups were additionally treated at neutral or lower pH prior toaddition to the cell layer.

FIG. 3 illustrates in vitro comparison of different coatingformulations, wherein Virus (WR.TK-Luc+) was enveloped with eitherPEG-FTS or DSPE-PEG containing lipid layers, or left uncoated. Virus wasthen either mixed with high dose VIG for 30 minutes, or left withoutantibody. The PEG-FTS enveloped virus then additionally had HCl added tolower the pH. Virus was then added to a HeLa cell layer, left for 24 hfor infection and gene expression to occur, before bioluminescence wasread. It was determined from FIG. 3 that use of a DSPE-PEG envelope(without VIG) resulted in about 50% recovery relative to naked virus.This result is a significant improvement over any similar reportedtechnologies with other viral backbones, where recovery of 1-5% istypical. Such results may are evidence of an advantage of encapsulatingthe MV form of vaccinia, to create a synthetic version of the EV, soretaining natural viral infection pathways.

However, when PEG-FTS was incorporated as a viral envelope, 100%recovery was achieved (after exposure to lower pH to degraft PEG)relative to naked virus. It is therefore apparent that combiningvaccinia MV form for encapsulation with pH-sensitive PEG degraftingtechnology resulted in complete recovery of the virus. Whereas exposureto VIG completely neutralized the naked virus, PEG-FTS coated virus wasprotected and around 50% infectious virus was recovered. This result wasobtained even though degrafting of PEG (through lowering of pH) occurredprior to layering of the particles (plus VIG) onto the cell layer forthe infection step.

In a subsequent set of representative experiments, naked or PEG-FTSenveloped virus were delivered intravenously (tail vein, 1e8 pfu/mouse,n=4 per group) to subcutaneous HCT 116 tumor-bearing athymic nude mice(see FIGS. 4A and 4B). In some groups, animal had previously received anintraperitoineal injection of VIG. In this way, it was possible toexamine delivery in the face of neutralizing antibody without dealingwith loss of tumor signal as a result of the CTL response targetinginfected tumor cells.

Unexpectedly, when synthetically enveloped virus (eVV) hereof wascompared to naked virus (VV) in the absence of VIG, the enveloped virusdisplayed significantly enhanced (p=0.009) viral gene expression fromthe tumor, indicating more efficient delivery. When similar comparisonswere made for a region of interest drawn over the upper body of themouse (including natural viral targets such as liver, spleen and lungs,but not the tumor), eVV displayed a 5-fold reduction in signal fromnormal tissues. This reduction is typical for successfully detargetedviral particles, but this usually occurs at the expense of infection ofthe tumor target. In the case of the enveloped viruses hereof, oneactually sees an increase in infection within the tumor.

When VIG was present, naked virus produced no detectable signal from thetumor. However enveloped virus was still capable of producing highlevels of tumor signal (although this was reduced around 3-fold relativeto enveloped virus delivery in naïve mice).

In another set of representative experiments, delivery and anti-tumoreffects were compared in naïve and fully immunized mice. Immunocompetentanimals were initially vaccinated with wild type vaccinia (strain WR, IP1e6 PFU) or left unvaccinated as controls. Vaccinated animals were leftfor 28 days to ensure virus was cleared and that the adaptive responsehad entered the memory phase, so as not to effect tumor implantation.Mice were then implanted with MC38 tumors subcutaneously. Once tumorsbecame palpable, mice were treated with a single intravenous injectionof 1e8 PFU of WR.TK− or enveloped WR.TK− (see FIGS. 5A and 5B).

As observed with the nude mouse model, it was seen that enveloped viruswas actually more efficient at infecting the tumor after systemicdelivery in naïve mice relative to naked virus. As expected, no viralgene expression was detected in the tumor after systemic delivery inpreviously immunized animals. Remarkably, enveloped virus producedgreater viral gene expression in the tumor after delivery in immunizedmice than naked virus achieved after delivery to naïve mice, this isdespite the expected anti-viral effects of the CTL response afterinfection of cells in the tumor.

The enhanced viral gene expression of the enveloped viruses hereof alsotranslated into enhanced therapeutic activity. Whereas naked virusproduced modest delays in tumor growth in this model in naïve mice, ithad no effect when delivered to previously immunized mice. Envelopedvirus hereof produced significantly enhanced therapeutic effects(relative to naked virus in naïve mice) whether it was delivered innaïve or pre-immunized mice. The enveloped viruses hereof may actuallyenhance anti-tumor effects relative to naked virus delivered in naïvemice, even when the enveloped virus hereof is delivered systemically infully immunized animals.

To more accurately model a clinical situation, where a patient may nothave been previously exposed to the virus, or may have been vaccinateddecades earlier with only limited immunity remaining, experiments wereperformed to repeat delivery of enveloped or naked virus to tumorbearing mice. Such experiments are complicated by the fact that mostsyngeneic tumors are highly aggressive and the time frame between tumorsbecoming palpable and the need to sacrifice the animal is often only 2-3weeks, about the same time as needed to raise a robust antibodyresponse.

In a number of experiments, BALB/c mice were implanted subcutaneouslywith RENCA tumors and then treated once the tumors became palpable viathe tail vein with 1e8 pfu of either naked or enveloped virus. After agap of either 3 days or 14 days a second round of the same therapy wasdelivered. Tumor burden was followed over time. It was seen in FIGS. 6Aand 6B that whereas repeat injections of naked virus provided noadditional therapeutic benefit relative to a single intravenousinjection (single injection tumor volume was 1720 mm³ at day 17 whenimaging was taken), enveloped virus produced significant additionaltherapeutic benefit after repeat cycles of systemic delivery.

FIG. 7 illustrates percent infection rate of several types of virusesenveloped or encapsulated under the methods hereof as compared to suchviruses without the envelopes hereof as controls. Improved results areobtained for representative viruses such as adenovirus (Ad) and HSV.These results indicate that other viral backbones (including both lipidand protein enveloped viruses) can be encapsulated within the syntheticlipid envelopes as described herein while retaining infectious capacity.The retained infections capacity is greater than that reported forpreviously described technologies (including, approaches to attach PEGto the viral surface to enhance systemic circulation and/or to detargetvirus from uptake in non-target tissues (such as the liver)). Theinfectious capacity, after addition of synthetic envelope for virusesthat do not naturally exist in different forms with different numbers oflipid layers (such as Adenovirus or HSV), may be reduced relative tothose viruses that do have this capability (such as vaccinia).

The HSV and adenovirus are commonly used as backbones for gene therapyor oncolytic virus therapies. Although there is some loss in infectivitywith these viruses, it is relatively small compared to previouslydescribed approaches.

Experimental

Cell lines and viral vectors: Tumor cell lines including 4T1 (mousebreast cancer); Renca (mouse renal cancer), HCT 116 (human colorectalcancer) and HeLa (human cervical cancer) were obtained from ATCC. MC38(mouse colorectal cancer) was obtained from David Bartlett, Universityof Pittsburgh. Cells were cultured as recommended. VacciniaImmunoglobulin (VIG) was a kind gift from CDC.

The vaccinia strain WR.TK-Luc+ contains an insertional mutation in theviral thymidine kinase (TK) gene, containing the luciferase transgeneunder control of the pSE/L promoter, and has been described previously.Virus was amplified in HeLa cells, lysed by freeze/thaw and purified byultracentrifuge banding and tangential flow.

Mice and mouse models. Mice (athymic nu-/nu-, BALB/c and C57/BL6) wereobtained from Charles River and were housed with food and water adlibitum. Tumors were formed through subcutaneous implantation of 1e6mouse tumor cells or 1e7 human tumor cells. Unless otherwise indicated,once tumor became palpable (50-100 mm³) animals were treated with 1e8PFU of virus or enveloped virus via tail vein injection. Subsequenttumor volume was determined by caliper measurement and viral geneexpression determined by bioluminescence imaging (on an IVIS200, PerkinElmer after IP delivery of D-luciferin substrate (Gold Bio)).

Synthesis of representative pH sensitive or responsive conjugates.Synthesis of several representative pH sensitive or responsiveconjugates suitable for formation of lipidic viral envelopes was carriedout. A series of pH-sensitive conjugates, containing two molecules ofFTS-H coupled to one molecule of PEG via a hydrazone linker areillustrated in FIG. 8. Conjugates with increasing carbon chain length orelectron withdrawing/contributing groups close to the hydrazone linkerwere studied to demonstrate their tunable pH-sensitivity (see FIG. 8).Synthesis of the four conjugates of FIG. 8 is straightforward, usingketone group on PEGylated molecules to react with FTS-H, which usuallycompleted within 2 hours. Successful synthesis was confirmed via ¹H NMRspectrums and MALDI-TOF of the conjugates.

Viral envelopes. Materials: Dimyristoyl phosphatidylcholine (DMPC),cholesterol (Chol)1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly ethyleneglycol)-2000] (DSPE-PEG2K), PEGSK-FTS-Hydrazide with hydrazine linker(PEGSK-FTS-H2).

Protocol: Dimyristoyl phosphatidylcholine (DMPC), cholesterol, andmPEG-FTS at a 2:1:0.1 molar ratio were dissolved in chloroform in aglass tube. The organic solvent was further removed by nitrogen flow toform a thin film. The film was dried under vacuum for 1 h to remove theremaining solvent. Virus was diluted with PBS, and then added to thetube to hydrate the thin film. The tube with the hydrated film wasplaced in an ultrasonic water bath (output control setting 4, Sonifer250) for 30 mins. The encapsulated virus was ready after stabilizing atroom temperature for 3 hours.

The foregoing description and accompanying drawings set forth a numberof representative embodiments at the present time. Variousmodifications, additions and alternative designs will, of course, becomeapparent to those skilled in the art in light of the foregoing teachingswithout departing from the scope hereof, which is indicated by thefollowing claims rather than by the foregoing description. All changesand variations that fall within the meaning and range of equivalency ofthe claims are to be embraced within their scope.

What is claimed is:
 1. A method of modifying a virus for in vivodelivery to a region of interest, comprising: forming an envelopingcomposition comprising a lipid conjugate formed by conjugating at leastone lipid with at least one hydrophilic compound via a linkage which iscleavable under conditions present in the region of interest, andcombining the virus with the enveloping composition to encompass thevirus within an enveloping structure.
 2. The method of claim 1 whereinthe enveloping composition forms a lipid bilayer to encompass the virus.3. The method of claim 2 wherein the enveloping composition comprises atleast one co-lipid.
 4. The method of claim 3 wherein the at least oneco-lipid is a phospholipid.
 5. The method of claim 3 wherein the atleast one lipid is selected from the group consisting of n-docosanoicacid, arachidic acid, stearic acid, palmitic acid, myristic acid, lauricacid, oleyl acid, vitamin E, embelin,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, or a compound havingthe formula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof.
 6. The method ofclaim 5 wherein the at least one lipid is selected from the groupconsisting of S-trans, trans-farnesylthiosalicylic acid, S-trans,trans-farnesylthiosalicylic acid amide (FTS-amide), S-trans,trans-farnesylthiosalicylic acid methylamide (FTS-MA) and S-trans,trans-farnesylthiosalicylic acid dimethylamide (FTS-DMA).
 7. The methodof any one of claims 1 through 6 wherein a family of the virus isselected from the group consisting of poxvidrae, denoviridae,herpesviridae, picomaviridae, rhabdoviridae, paramyxoviridae,retroviridae, togaviridae or reoviridae.
 8. The method of any one ofclaims 1 through 6 wherein the virus is selected from the groupconsisting of a vaccinia virus, a myxoma virus, an avipox virus, anadenovirus, a herpes simplex virus (HSV) coxsackie virus, a vesicularstomatitis virus (VSV), a Newcastle disease virus (NDV), anadeno-associated virus (AAV), a polio virus, a lenti virus, aretrovirus, a reovirus, or a sindbis virus.
 9. The method of claim 7wherein the family of the virus is poxvidrae.
 10. The method of claim 9wherein the virus is a vaccinia virus.
 11. The method of claim 9 whereinthe virus is a mature vaccinia virus.
 12. The method of any one ofclaims 1 through 6 wherein the region of interest comprises a tumor andthe virus is modified to treat the tumor.
 13. The method of any one ofclaims 1 through 6 wherein the at least one lipid isfarnesylthiosalicylic acid or a farnesylthiosalicylic acid amide. 14.The method of any one of claim 1 through 6 wherein the at least onehydrophilic compound comprises at least one hydrophilic oligomer or atleast one hydrophilic polymer.
 15. The method of claim 14 wherein the atleast one hydrophilic oligomer or the at least one hydrophilic polymeris selected from the group consisting of a polyalkylene oxide, apolyvinylalcohol, a polyacrylic acid, a polyacrylamide, a polyoxazoline,a polysaccharide and a polypeptide.
 16. The method of claim 14 whereinthe at least one hydrophilic compound is a polyalkylene oxide.
 17. Themethod of claim 16 wherein the polyalkylene oxide is a polyethyleneglycol.
 18. The method of claim 17 wherein the polyethylene glycol has amolecular weight of at least 1 KDa.
 19. The method of any one of claims1 through 6 wherein the linkage is sensitive to pH.
 20. The method ofclaim 19 wherein the linkage comprises a hydrazine group.
 21. The methodof claim 3 further comprising including an additive within theenveloping composition to control stability of the enveloping structure.22. The method of claim 21 wherein the additive is cholesterol or1,2-dioleoyl-sn-glycero-3-phosphoethanolamine.
 23. A formulation for invivo delivery to a region of interest, comprising: a virus; a syntheticenveloping composition encompassing the virus and comprising a lipidconjugate formed by conjugating at least one lipid with at least onehydrophilic compound via a linkage which is cleavable under conditionspresent in the region of interest.
 24. The formulation of claim 23wherein the enveloping composition forms a lipid bilayer encompassingthe virus.
 25. The formulation of claim 24 wherein the envelopingcomposition comprises at least one co-lipid.
 26. The formulation ofclaim 25 wherein the at least one co-lipid is a phospholipid.
 27. Theformulation of claim 25 wherein the at least one lipid is selected fromthe group consisting of n-docosanoic acid, arachidic acid, stearic acid,palmitic acid, myristic acid, lauric acid, oleyl acid, vitamin E,embelin, 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, or acompound having the formula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof.
 28. The formulationof claim 27 wherein the at least one lipid is selected from the groupconsisting of S-trans, trans-farnesylthiosalicylic acid, S-trans,trans-farnesylthiosalicylic acid amide (FTS-amide), S-trans,trans-farnesylthiosalicylic acid methylamide (FTS-MA) and S-trans,trans-farnesylthiosalicylic acid dimethylamide (FTS-DMA).
 29. Theformulation of any one of claims 23 through 28 wherein a family of thevirus is selected from the group consisting of poxvidrae, denoviridae,herpesviridae, picornaviridae, rhabdoviridae, paramyxoviridae,retroviridae, togaviridae or reoviridae.
 30. The formulation of any oneof claims 23 through 28 wherein the virus is selected from the groupconsisting of a vaccinia virus, a myxoma virus, an avipox virus, anadenovirus, a herpes simplex virus (HSV) coxsackie virus, a vesicularstomatitis virus (VSV), a Newcastle disease virus (NDV), anadeno-associated virus (AAV), a polio virus, a lenti virus, aretrovirus, a reovirus, or a sindbis virus.
 31. The formulation of claim29 wherein the family of the virus is selected from the group consistingof poxvidrae
 32. The formulation of claim 30 wherein the virus is avaccinia virus.
 33. The formulation of claim 30 wherein the virus is amature vaccinia virus.
 34. The formulation of any one of claims 23through 28 wherein the region of interest comprises a tumor and thevirus is modified to treat the tumor.
 35. The formulation of any one ofclaims 23 through 28 wherein the at least one lipid isfarnesylthiosalicylic acid or a farnesylthiosalicylic acid amide. 36.The formulation of any one of claim 23 through 28 wherein the at leastone hydrophilic compound comprises at least one hydrophilic oligomer orat least one hydrophilic polymer.
 37. The formulation of claim 36wherein the at least one hydrophilic oligomer or the at least onehydrophilic polymer is selected from the group consisting of apolyalkylene oxide, a polyvinylalcohol, a polyacrylic acid, apolyacrylamide, a polyoxazoline, a polysaccharide and a polypeptide. 38.The formulation claim 37 wherein the at least one hydrophilic compoundis a polyalkylene oxide.
 39. The formulation of claim 38 wherein thepolyalkylene oxide is a polyethylene glycol.
 40. The formulation ofclaim 29 wherein the polyethylene glycol has a molecular weight of atleast 1 KDa.
 41. The formulation of any one of claims 23 through 28wherein the linkage is sensitive to pH.
 42. The formulation of claim 41wherein the linkage comprises a hydrazine group.
 43. The formulation ofclaim 26 further comprising an additive.
 44. The formulation of claim 45wherein the additive is cholesterol.
 45. A method of in vivo delivery ofa virus to a region of interest, comprising: injection of a formulationof any one of claims 23 through
 47. 46. A method of modifying a virusfor in vivo delivery to a region of interest, comprising: forming anenveloping composition comprising a lipid having the formula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof; and combining thevirus with the enveloping composition to encompass the virus.
 47. Themethod of claim 48 wherein the enveloping composition forms a lipidbilayer.
 48. The method of claim 48 wherein the enveloping compositioncomprises at least one co-lipid.
 49. The method of claim 50 wherein theat least one co-lipid is a phospholipid.
 50. The method of claim 50wherein lipid is selected from the group consisting of S-trans,trans-farnesylthiosalicylic acid, S-trans, trans-farnesylthiosalicylicacid amide (FTS-amide), S-trans, trans-farnesylthiosalicylic acidmethylamide (FTS-MA) and S-trans, trans-farnesylthiosalicylic aciddimethylamide (FTS-DMA).
 51. A formulation for in vivo delivery to aregion of interest, comprising: a virus; a synthetic envelopingcomposition encompassing the virus and comprising a lipid having theformula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof.
 52. A compositionformed by conjugating at least one lipid with at least one hydrophiliccompound via a pH-sensitive hydrazine linkage which is cleavable underconditions present in a region of interest.
 53. The composition of claim54 wherein the composition forms a lipid bilayer.
 54. The composition ofclaim 55 wherein the enveloping composition comprises at least oneco-lipid.
 55. The composition of claim 56 wherein the at least oneco-lipid is a phospholipid.
 56. The composition of claim 56 wherein theat least one lipid is selected from the group consisting of n-docosanoicacid, arachidic acid, stearic acid, palmitic acid, myristic acid, lauricacid, oleyl acid, vitamin E, embelin,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, or a compound havingthe formula:

wherein R₁ is a farnesyl group, a geranyl group or geranyl-geranylgroup, X is O, S, SO, SO₂, NH or Se, Z is C—R₂ or N, R₂ is H, CN, CO₂R₇,SO₃R₇, CONR₇R₈ or SO₂NR₇R₈, wherein R₇ and R₈ are each independently H,an alkyl group, an alkenyl group, CO₂M or SO₃M, wherein M is a cationand R₃, R₄, and R₅ are independently H, a carboxyl group, an alkylgroup, an alkenyl group, an aminoalkyl group, a nitroalkyl group, anitro group, a halo atom, an amino group, a mono-alkylamino group, adi-alkylamino group, mercapto group, a mercaptoalkyl group, an azidogroup or a thiocyanato group, or derivative thereof.
 57. The compositionof claim 58 wherein the at least one lipid is selected from the groupconsisting of S-trans, trans-farnesylthiosalicylic acid, S-trans,trans-farnesylthiosalicylic acid amide (FTS-amide), S-trans,trans-farnesylthiosalicylic acid methylamide (FTS-MA) and S-trans,trans-farnesylthiosalicylic acid dimethylamide (FTS-DMA).
 58. Thecomposition of any one of claims 54 through 59 wherein the at least onelipid is farnesylthiosalicylic acid or a farnesylthiosalicylic acidamide.
 59. The composition of any one of claim 54 through 59 wherein theat least one hydrophilic compound comprises at least one hydrophilicoligomer or at least one hydrophilic polymer.
 60. The composition ofclaim 61 wherein the at least one hydrophilic oligomer or the at leastone hydrophilic polymer is selected from the group consisting of apolyalkylene oxide, a polyvinylalcohol, a polyacrylic acid, apolyacrylamide, a polyoxazoline, a polysaccharide and a polypeptide. 61.The composition of claim 62 wherein the at least one hydrophiliccompound is a polyalkylene oxide.
 62. The composition of claim 63wherein the polyalkylene oxide is a polyethylene glycol.
 63. Thecomposition of claim 64 wherein the polyethylene glycol has a molecularweight of at least 1 KDa.
 64. The composition of any one of claims 54through 59 wherein the linkage is sensitive to pH.